Development of suitable, if not ideal scaffold for the regeneration of tissue is the focus of substantial research efforts. Producing a scaffold easily and inexpensively from common materials that is easy to use and that supports correct cell differentiation and growth is no trivial matter. As one example, bone has natural healing capacity but trauma due to sports or other accidents cause injuries beyond natural healing capacity. Bone cancer in children accounts for 5% of total cancers. Recent advances in biomaterials for bone regeneration have led to materials, mostly with composite or mixture of scaffolds with hydroxyapatite or other calcium phosphate nanoparticles. At best, bioactivity of the materials has been demonstrated to the induction of osteogenicity due to the degradation products of the composite scaffolds. It has been known since long that most tissues in our body has multi-scale hierarchy. Bone is formed by the deposit of inorganic minerals in an organic matrix, forming organic-inorganic nanocomposites. With clearer understanding of bone extracellular matrix (ECM) and its components, bottom-up assembly process of bone development is well studied. Bioactivity of natural bone ECM is found to be majorly due to collagen and non-collagenous proteins (NCPs). The interaction between organic matrix and minerals is considered to be the most important feature of mineralization process.
Bone loss is currently managed by filling the damaged tissue with non-bioactive and non-biodegradable materials such as metals and ceramics. They are ideal strategies for short-term management of the injury as they provide good mechanical support to maintain overall function. However, they lead to fibrotic tissue formation in the surrounding native bone due to mechanical mismatch. Moreover, different grafts are widely used for managing bone loss in clinic, however, they are limited due to complications such as graft rejection and disease transmission. The best strategy for promoting bone regeneration should be to learn from native bone development and trying to incorporate required features. Bone is composite material of organic and inorganic components. More specifically, two third of dried bone tissue in vertebrates is inorganic and the remaining one third part is composed of organic materials. Cells constitute only a small portion of total organic mass, in which collagen constitutes almost 85-90% of total organic mass. There are other components in extracellular matrix such as, acidic proteins, proteoglycans, phosphoproteins, glycoproteins and sialoproteins. Bone extracellular matrix has multiple components organized anisotropically. Characteristic self-assembly of amino acids forms triple helical structure called tropocollagen. Such self-assembly at atomic scale facilitates organization of collagen type I fibrils with overlapping and hole regions which appear as dark and light bands when observed under transmission electron microscopy (See Traub, W., T. Arad, and S. Weiner, Origin of Mineral Crystal Growth in Collagen Fibrils. Matrix, 1992. 12(4): p. 251-255).
Collagen is acknowledged as a key determinant which dictates mineralization by controlling microenvironment, mainly via charge interaction with mineral phase (See Nudelman, F., et al., In vitro models of collagen biomineralization. Journal of Structural Biology, 2013. 183(2): p. 258-269). There are several evidences (See Traub, W., T. Arad, and S. Weiner, Origin of Mineral Crystal Growth in Collagen Fibrils. Matrix, 1992. 12(4): p. 251-255; Silver. F. H. et al., Molecular Basis for Elastic Energy Storage in Mineralized Tendon, Biomacromolecules, 2001. 2(3): p. 750-756; Silver, F. H. and W. J. Landis, Deposition of apatite in mineralizing vertebrate extracellular matrices: A model of possible nucleation sites on type I collagen, Connective Tissue Research, 2011. 52(3): p. 242-254; Dahl, T., B. Sabsay, and A. Veis, Type I Collagen-Phosphophoryn Interactions: Specificity of the Monomer-Monomer Binding. Journal of Structural Biology, 1998. 123(2): p. 162-168; and Landis, W. J. and F. H. Silver, The structure and function of normally mineralizing avian tendons, Comp Biochem Physiol A Mol Integr Physiol, 2002. 133(4): p. 1135-57) which suggest that side chains of positively and negatively charged amino acids present in the light band (gap region) could provide a three dimensional microenvironment to initiate nucleation of apatite by spatially guiding calcium and phosphate ions (See Dahl, T., B. Sabsay, and A. Veis, Type I Collagen-Phosphophoryn Interactions: Specificity of the Monomer-Monomer Binding. Journal of Structural Biology, 1998. 123(2): p. 162-168; Chapman, J. A. and R. A. Hardcastle, The Staining Pattern of Collagen Fibrils: Ii. a Comparison With Patterns Computer-Generated From the Amino Acid Sequence. Connective Tissue Research, 1974. 2(2): p. 151-159; and Chapman, J. A., The Staining Pattern of Collagen Fibrils: I. an Analysis of Electron Micrographs. Connective Tissue Research, 1974. 2(2): p. 137-150). However, the precise interaction nature of collagen-mineral interaction is far from clearly understood (See Landis, W. J. and F. H. Silver, Mineral deposition in the extracellular matrices of vertebrate tissues: identification of possible apatite nucleation sites on type I collagen. Cells Tissues Organs, 2009. 189(1-4): p. 20-4). Moreover, there are other in vitro studies which suggest failure of collagen alone to interact with minerals and to create nucleation sites (See Stetler-Stevenson, W. G. and A. Veis, Bovine dentin phosphophoryn: calcium ion binding properties of a high molecular weight preparation. Calcif Tissue Int. 1987. 40(2): p. 97-102; and Stetler-Stevenson, W. G. and A. Veis, Type I collagen shows a specific binding affinity for bovine dentin phosphophoryn. Calcif Tissue Int, 1986. 38(3): p. 135-41). Additionally, conclusion of some studies advocate about NCPs (See Dahl, T., B. Sabsay, and A. Veis, Type I Collagen-Phosphophoryn Interactions: Specificity of the Monomer-Monomer Binding. Journal of Structural Biology, 1998. 123(2): p. 162-168; Stetler-Stevenson, W. G. and A. Veis, Bovine dentin phosphophoryn: calcium ion binding properties of a high molecular weight preparation. Calcif Tissue Int, 1987. 40(2): p. 97-102; Stetler-Stevenson, W. G. and A. Veis, Type I collagen shows a specific binding affinity for bovine dentin phosphophoryn. Calcif Tissue Int, 1986. 38(3): p. 135-41; and Ganss, B., R. H. Kim, and J. Sodek, Bone sialoprotein. Crit Rev Oral Biol Med, 1999. 10(1): p. 79-98) as mediators in nucleating apatite not only in gap region but also in overlap region mainly (See Deshpande. A. S. and E. Beniash, Bio-inspired Synthesis of Mineralized Collagen Fibrils. Cryst Growth Des, 2008. 8(8): p. 3084-3090). These NCPs nucleate mineral deposition with the help of interaction of functional group with calcium and phosphate ions. Therefore, structural features of collagen, either alone or along with other physicochemical factors exerted by non-collagenous proteins (NCPs) in the matrix, guide and control mineralization.
In addition to increasing interest in developing biomimetic materials, the advancement in mineralization knowledge has righteously set the new goal that the ideal material for bone regeneration will induce the same response in the body as natural bone matrix. It is important for any material intended for promoting mineralization that it develops nucleation sites similar to collagen or NCPs or both. It should also incorporate bioactive or functional molecular template which can selectively induce nucleation and provide hydrogel-like environment in which minerals can grow (See Nudelman, F. and N. A. J. M. Sommerdijk, Biomineralization as an Inspiration for Materials Chemistry. Angewandte Chemie-International Edition, 2012. 51(27): p. 6582-6596). Thus, we took up bottom up approach by designing nano-scale building blocks to shape into complex, multi-scale hierarchical structures with desired functional features for promoting mineralization. In order to maximize bioactivity of our scaffolds we strategized to incorporate both collagen stereochemistry and functional role of NCPs in nucleation and growth. Collagen-mimetic structure will provide similar structural features as steric arrangement of amino acids of tropocollagen and its charges. Such structural mimicry provides hierarchy at different length-scale, to provide the desired function in case of collagen. Along with collagen-mimetic structural features, incorporation of carboxylic and sulfate functional groups will amplify bioactivity in similar way as in NCPs and other ECM components which are also proven to be functioning as nucleators.
Regenerative approaches that employ extracellular matrix (ECM)-like materials have been explored by researchers in the last decade, however, they fail to regenerate and remodel the bone. Natural bone development mechanisms need to be facilitated in regenerative materials based therapies in order to promote proper bone regeneration (See Nudelman, F. and N. A. J. M. Sommerdijk. Biomineralization as an Inspiration for Materials Chemistry. Angewandte Chemie-International Edition, 2012. 51(27): p. 6582-6596). Many approaches employ inorganic-organic composites, a blend of synthetic minerals and natural or synthetic scaffolds which often end up using growth factors to promote bone growth due to lack of bioactivity from scaffolds.